Pathogens and histopathological characteristics of shrimp postlarvae bacterial vitrified syndrome (BVS) in the Litopenaeus vannamei
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摘要: 为进一步查明对虾“玻璃苗”的主要致病原，实验通过对河北省沧州市凡纳滨对虾苗种玻化症进行了流行病学调查及病原、病理分析。结果发现，患病虾苗表现为活力降低、厌食直至空肠、空胃，虾体消瘦、暗浊；肝胰腺组织坏死性萎缩、轮廓模糊、颜色变浅呈淡黄色，甚至肝胰腺区由正常的饱满褐色组织变为无组织结构的“玻璃化”状态。组织病理观察结果显示，患病对虾肝小管上皮细胞坏死、脱落，肝小管中充斥大量的碎片组织，并逐步褐化、黑化，甚至肝小管组织大面积坏死，留有连片玻璃样均质化区域；肠道内充斥大量的组织碎片，绒毛膜脱落消失。超微组织病理观察发现，患病对虾肝小管上皮细胞的细胞膜消融，细胞器解体，细胞核固化；其后细胞解体、脱落，甚至肝小管组织结构解体消融；肝胰腺、肠道、胃黏膜周围发现大量细菌，优势菌株为杆状菌且呈弧形，未发现病毒粒子的存在。从患病虾苗分离出2株优势菌(Lv-A和Lv-B)，经人工浸染实验发现，Lv-A和Lv-B可致凡纳滨对虾PL7苗种出现与自然患病相同的玻璃化症状，其半致死浓度分别为1.62×103和5.38×103 CFU/mL，致病力强。根据16S rDNA和gyrB序列分析结果发现，Lv-A和Lv-B与溶藻弧菌、新喀里多尼亚弧菌和副溶血弧菌均有较高相似性。初步将该病命名为虾苗细菌性玻化症(shrimp postlarva bacterial vitrified syndrome，BVS)。药敏实验结果显示，Lv-A和Lv-B均对米诺环素、多西环素、萘啶酸等敏感，而对新霉素、吡哌酸、利福平等耐药。本研究为BVS的有效防控、保障对虾行业健康发展提供理论基础和技术支撑。Abstract: The shrimp postlarva vitrified syndrome broke out in spring of 2020 and spread explosively along the coastal areas from south to north of China. In order to find out the main pathogenic agents of shrimp postlarva vitrified syndrome, in this study, the pathogen was isolated and identified, and the histopathology was investigated. The postlarva symptoms included emaciation, dark cloud, decreased activity, anorexia, empty intestinal tract and stomach. The hepatopancreas showed atrophy, blurring of contour, paleness and even vitrified syndrome. Histopathological analysis showed that the epithelial cells of liver tubule were necrotic and exfoliated, liver tubule was filled with a large amount of debris tissue, leaving continuous glassy homogeneous areas. The intestinal tract was filled with tissue fragments, chorionic membrane fell off and even disappears. Ultrastructural pathological examination showed that the membranes of epithelial cells were ablated, organelles disintegrated, and nuclei were solidified. Subsequently, the cells disintegrated, fell off, and even the hepatic tubular tissue structure was ablated. The bacteria were found in the hepatopancreas, intestinal tract and gastric mucosa. The dominant strain was rod-shaped and curved, and no virions were found. Two dominant bacteria (Lv-A, Lv-B) were isolated from the diseased shrimp postlarvae. Artificial infection experiments illustrated that the strains of Lv-A and Lv-B were the causative pathogens with a median lethal dose of 1.62×103 CFU/mL and 5.38×103 CFU/mL, respectively. Based on molecular analyses (16S rDNA and gyrB), Lv-A and Lv-B were highly similar to Vibrio alginolyticus, V. neocaledonicus and Vibrio parahaemolyticus, preliminarily named shrimp postlarva bacterial vitrified syndrome (BVS). The chemotherapeutant sensitivity tests illustrated that Lv-A and Lv-B were sensitive to minocycline, doxycycline, nalidixic acid, etc. and resistant to neomycin, pipemidic, rifampicin, etc. This study provides theoretical basis and technical support for the effective prevention of BVS and the healthy development of shrimp industry.
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图 图版Ⅰ 凡纳滨对虾虾苗细菌性玻化症症状及菌株Lv-A和Lv-B的形态观察
Figure 图版Ⅰ. Bacterial vitrified syndrome of shrimp postlarva and morphology of strains Lv-A and Lv-B
图 1 基于16S rDNA(a)和gyrB (b)的菌株Lv-A和Lv-B系统进化树
Figure 1. Phylogenetic trees of Lv-A and Lv-B based on 16S rDNA (a) and gyrB (b)
图 图版Ⅱ 患病凡纳滨对虾虾苗组织病理变化
Figure 图版Ⅱ. The pathological changes of L. vannamei larvae associated with bacterial infection
图 图版Ⅲ 患病凡纳滨对虾虾苗超微组织病理特征
Figure 图版Ⅲ. The ultrastructural pathology characteristics of L. vannamei larvae associated with bacterial infection
表 1 菌株Lv-A和Lv-B对凡纳滨对虾虾苗的人工感染实验结果
Table 1. Results of artificial challenge to shrimp postlarvae using Lv-A and Lv-B
组别
group攻毒浓度/(CFU/mL)
infection concentration尾数/尾
fish number攻毒不同时间的死亡数量/尾
number of fish deaths after challenge at different time累计死亡数/尾
cumulative deaths12 h 24 h 36 h 48 h 60 h 72 h Lv-A 1.02×106 30 0 29 1 0 0 0 30 1.02×105 30 0 20 1 7 2 0 30 1.02×104 30 0 2 9 10 3 0 24 1.02×103 30 0 1 0 7 1 1 10 1.02×102 30 0 2 0 1 0 0 3 1.02×101 30 0 0 0 1 0 0 1 Lv-B 1.30×106 30 0 5 20 1 4 0 30 1.30×105 30 0 0 0 10 8 4 22 1.30×104 30 0 1 3 3 5 8 20 1.30×103 30 0 0 0 2 2 3 7 1.30×102 30 0 1 2 0 1 1 5 1.30×101 30 0 0 0 1 0 1 2 对照 control 1.5%NaCl 30 0 0 1 0 0 0 1 
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表 2 菌株Lv-A和Lv-B鉴定结果
Table 2. Results of the identification of Lv-A and Lv-B
基因名称
gene数据库
database菌株 strains Lv-A Lv-B 16S rDNA Blast 序列大小/bp sequence size 1 411 1 409 最大相似菌株 most similar strain 溶藻弧菌 V. alginolyticus 溶藻弧菌 V. alginolyticus 覆盖度/% coverage 100 100 相似度/% identity 99.79 99.86 EzBio 最大相似菌株 most similar strain 新喀尼亚弧菌 V. neocaledonicus 溶藻弧菌 V. alginolyticus 覆盖度/% coverage 95.8 95.6 相似度/% identity 99.64 99.43 gyrB Blast 序列大小/bp sequence size 1 166 1 159 最大相似菌株 most similar strain 副溶血弧菌 V. parahaemolyticus 副溶血弧菌 V. parahaemolyticus 覆盖度/% coverage 98 98 相似度/% identity 99.22 99.30
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表 3 菌株Lv-A和Lv-B对抗生素的敏感性
Table 3. The drug sensitivity of Lv-A and Lv-B to different kinds of antibiotics
药物名称
antibiotics药物含量/(μg/片)
contents抑菌圈直径/mm
diameter of inhibition敏感度
sensitivityLv-A Lv-B Lv-A Lv-B 红霉素 erythromycin 15 12 15 R I 米诺环素 minocycline 30 24 25 S S 多西环素 doxycyclin 30 19 21 S S 四环素 tetracycline 30 14 13 I I 新霉素 neomycin 30 12 12 R R 吡哌酸 pipemidic 30 15 17 R R 萘啶酸 nalidixic acid 30 22 27 S S 阿奇霉素 azithromycin 15 14 22 I S 克拉霉素 clarithromycin 15 15 17 I I 乙酰螺旋霉素 acetylspiramycin 30 0 0 R R 链霉素 streptomycin 10 0 0 R R 卡那霉素 kanamycin 30 18 0 S R 庆大霉素 gentamycin 10 12 14 R I 丁胺卡那 amikacin 30 13 14 R I 头孢哌酸 salbactam acid 75/75 22 18 S I 利福平 rifampicin 5 12 12 R R 呋喃唑酮 furazolidone 30 15 11 I R 复方新诺明 paediatric compound sulfamethoxazole tablets 23.75/1.25 0 0 R R 新生霉素 novobiocin 30 18 23 I S 多粘菌素B bpolymyxin B 300IU 10 0 R R 氟罗沙星 fleroxacin 5 22 27 S S 诺美沙星 lomefloxacin 10 14 22 R S 环丙沙星 ciprofloxacin 5 19 25 I S 氧氟沙星 ofloxacin 5 21 23 S S 诺氟沙星 norfloxacin 10 17 20 S S 头孢唑肟 ceftizoxime 30 15 20 R R 头孢噻肟 cefotaxime 30 17 21 R R 头孢曲松 ceftriaxone 30 18 20 R I 头孢他啶 ceftazidime 30 13 10 R R 头孢拉定 cefradine 30 0 0 R R 青霉素 penicillin 10 0 0 R R 苯唑西林 oxacillin 1 0 0 R R 氨苄西林 ampicillin 10 0 0 R R 头孢氨苄 cefalexin 30 0 0 R R 头孢唑啉 cefazolin 30 0 10 R R 氯霉素 chloromycetin 30 16 12 I R 恩诺沙星 enrofloxacin 10 16 21 I S 氟苯尼考 florfenicol 30 16 21 I S 注：R. 耐药；I. 居中；S. 敏感
Notes: R. resistance; I. intermediate; S. sensitivity
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